The principle aim of the work proposed here is to determine which step or step(s) of the nucleotide triphosphate hydrolysis mechanism limits shortening velocity in muscle. Recent work in my laboratory has shown that ADP dissociation from actomyosin-S1 is sufficiently slow to be the molecular step that limits unloaded shortening in mammalian muscle. To further test this hypothesis the rate constants of the dissociation of a series of nucleoside disphosphates (NDPs) from actomyosin-S1, will be measured using the stopped flow light scattering method. We have shown in preliminary studies that the complementary series of nucleoside triphosphates (NTPs) differ widely in their ability to support shortening and motility as measured in skinned muscle fibers and using the in vitro motility assay of Kron and Spudich. NTPs that poorly support contraction and motility, GTP and lTP, show novel steady state kinetics in which the rate of hydrolysis is strongly inhibited at moderately high actin concentrations. This indicates that one or more steps of the mechanism between attached crossbridge state is much slower for NTPs that support contraction poorly than for NTPs that support contraction well. It therefore appears that a sufficiently rapid rate of a critical attached crossbridge step is essential support normal contraction. We propose to use the methods of stopped-flow fluorescence, rapid chemical quenching, and intermediate exchange to determine which step or steps of the actomyosin hydrolysis mechanisms are changed for a series of NTPs. Altering the structure of the nucleotide substrates provides a critical test of which step(s) of the actomyosin NTP hydrolysis mechanism are essential for contraction and/or limit shortening velocity. An increased understanding of mechanism by which the chemical energy of nucleotide triphosphate hydrolysis is coupled to the production of mechanical work in muscle will improve our understanding of the molecular mechanisms that underly some of the alterations in contractility observed in heart disease and other muscle disorders.